Microbial degradation of obnoxious organic wastes into innocuous materials

ABSTRACT

This invention relates to microbial methods and materials useful in the degradation of organic chemicals having toxic and obnoxious characteristics into innocuous materials compatible with the environment and to the process comprising identification, production and utilization of microorganisms for said purposes.

This application is a division of application Ser. No. 305,079, filedSept. 24, 1981.

BACKGROUND OF THE INVENTION

The unprecedented growth of the chemical industry since World War II hasled to somewhat over 35 million metric tons of mostly toxic waste beinggenerated. Large quantities of synthetic halogenated materials such asdielictric fluids, flame retardants, refrigerants, heat transfer fluids,lubricants, protective coatings, pesticides, including herbicides andinsecticides, and many other chemicals and petroleum products useful inagriculture, industry and health care have been manufactured and used tothe benefit of mankind. In many cases, these materials and theirby-products or residues from their manufacture have been released intothe ecosphere and have been accumulated in landfills, the atmosphere,lakes, rivers and streams, as runoff or direct discharge.

Many of the halogenated chemicals employed in these applications inagriculture and industry are toxic and accumulate in animal and planttissues causing serious discomfort or health problems. Many also persistin the environment because they are not biodegradable because of theinability of natural microflora generally available in the environmentto degrade them.

Many methods and techniques have been proposed and used for disposing ofand/or treating these chemicals, their by-products, and their wastes ina way which makes them compatible with the environment. In spite of allthe effort and money being spent to clean up the ecosphere, the problempersists. Disposing of waste chemicals accumulated from past practicesand preventing future accumulation of such noxious materials is ofworldwide concern. The continuing manufacture of such chemicals, whichhave proven to be essential and so important and necessary toagriculture, industry, and health care in the betterment of mankindcontinues, and so does the piling up of the obnoxious wastes which arenon-biodegradable or not disposable into the natural carbon cycle.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to methods for collecting, making, and usingmicroorganisms capable of dissimilating chemicals such as halogenatedorganic compounds back into the natural carbon cycle.

In addition, this invention relates to novel plasmids, or unique regionsof the bacterial chromosome associated with the degradative activities,their microbiological preparation and to their utility as cloningvehicles in recombinant DNA work especially for the purpose of enhancingthe ability of microorganisms to biodegrade obnoxious halogenatedorganic wastes.

More particularly, this invention relates to bacterial strains orcultures capable of converting chlorinated aromatic compounds intocarbon dioxide, water and salt.

Further, this invention relates to the process for microbial degradationof obnoxious organic wastes into innocuous materials which comprises:

1--collecting a sample of material from the site contaminated with theobnoxious chemicals,

2--enriching the microorganisms found living in the sample,

3--separating the strains of microorganisms capable of having differentmetabolisms for the various chemicals in the sample from the site, fromeach other,

4--purifying the strains which are capable of biodegrading the chemicalsto be disposed of,

5--applying the strain(s) to the locus of the contaminants to bedisposed of, and

6--monitoring of removal of the contaminants at the locus of theapplication.

Still further, this invention relates to the process for improving thebiodegradability of the biologically purified cultures of themicroorganisms isolated from the locus of the contaminants (the purifiedbacterial strains) which comprises transforming the purified strains toanother strain of bacteria capable of metabolizing a different chemicalin the contaminants to be disposed of.

Another process of this invention comprises conjugal mating the purifiedstrains as the genetic donor with a bacterial strain capable ofmetabolizing a different chemical in the contaminants to be disposed.

Still another process of this invention involves cell fusion whereby twobacteria become one and new combinations of genes are obtained.

Another process of this invention comprises transduction wherebybacterial viruses transduce genetic material from a donor bacteria to agenetic recipient.

THE PRIOR ART

Over the past few decades especially in the last ten years, hundreds ofmillions of dollars and many years of effort have been spent in tryingto clean up the ecosphere. There have been many academic disclosureswhich describe bacteria which grow on aliphatic, cycloaliphatic,aromatic and polynuclear aromatic compounds. For example, a variety ofmicroorganisms have been isolated that have the capability ofefficiently utilizing aromatic organic chemicals as sole carbon sourcesfor growth (e.g. toluene, phenol, and naphthalene). See Clarke, P. H.and Ornston, L. N. (1975) "Metabolic Pathways and Regulations", 1,191-196 in Clarke, P. H. and Richmond, M. H. (ed.), "Genetics andBiochemistry of Pseudomonas", John Wiley, London. However, thecorresponding chlorinated aromatic compounds (chlorotoluenes,chlorophenols, chloronaphthalenes) are biodegraded very slowly, if atall. See Alexander, M. (1973) "Non-Biodegradable and Other RecalcitrantMolecules", Biotechnology--Bioengineering, 15: 611-647. Notwithstanding,micro-organisms have been isolated from the environment that are capableof growth on chlorinated aromatic compounds. For example, Chakrabarty,A. M., (1976) "Plasmids in Pseudomonas"; Ann. Rev. Genet. 10, 7-30,discloses bacteria which utilize haloaromatic compounds and thedegradative pathways of intermediates involved. Several otherpublications deal with the microbiodegradation of halogenatedhydrocarbons. For example, Bourquin, A. W. and Gibson, D. T. (1978)"Microbial Degradation of Halogenated Hydrocarbons; Water ChlorinationEnvironmental Impact and Health Effects", 2, 253-264 disclose variousmicroorganisms such as: Aspergillus sp., Achromobacter sp., Arthrobactersp. and Clostridium sp., as useful for dehalogenation of varioussubstrates such as 2-chlorophenoxyacetate, 2,4-dichlorophenol,3-chlorobenzoate, hexachlorocyclohexane, and 4-chlorobenzoate. Gibson,D. T., Koch, J. R., Schuld, C. L. and Kallio, R. E.(1968)--Biochemistry, 7 No. 11 3795-3802 in their paper on OxidativeDegradation of Aromatic Hydrocarbons by Microorganisms including theMetabolism of Halogenated Aromatic Hydrocarbons disclosed Pseudomonasputida as useful in the degradation of toluene and chlorinated compoundssuch as halobenzenes and p-chlorotoluene and state that the presence ofhalogen atoms greatly reduces the biodegradability of aromaticcompounds. They also disclose that microorganisms have also beenisolated that have the capability to co-metabolize a chlorinatedaromatic chemical during growth on its nonchlorinated analog. Forexample, the conversion of chlorotoluene to chlorocatechol during growthof Pseudomonas putida on toluene has been demonstrated. This organismwould not further metabolize the chlorocatechol, rather it is known thatother microorganisms possess the ability to metabolize chlorocatechols,see Dorn, E. M., Hellwig and Reineke, W. and Knackumss, H. J. (1974),"Isolation and Characterization of a 3-Chlorobenzoate DegradingPseudomonas", Arch. Microbiology 99, 61-70 and also see Evans, W. C.;Smith, B. S. W.; Fernley, H. N.; and Davies, J. I. (1971), "BacterialMetabolism of 2,4-Dichlorophenoxy Acetate, Biochem J., 122, 543-551.Chlorocatechol is known to be an intermediate in many of the metabolicpathways for utilization of chlorinated aromatic compounds. Thechlorocatechol is further metabolized with the subsequent removal ofchlorine. See Tiedje, J. M.; Duxbury, J. M.; Alexander, M. and Dawson,J. E. (1969), 2,4 D Co-metabolism: Pathway of Degradation ofChlorocatechols by Arthrobacter, J. Agr. Food Chem, 17, 1021-1026.Hartmann, J., Reineke, W., Knackmuss, H. J., (1979) Applied &Environmental Microbiology: 37, No. 3, 421-428 show a species ofPseudomonas identified as sp. WR912 capable of degrading chlorobenzoicacids. Shubert, R., (1979) Fed. Ministry for Research and Technology,Goethe University, Frankfurt, W. Germany in his paper on Toxicity ofOrganohalogen Compounds, discloses the Minimal Inhibitory Concentrationspreventing growth of various bacteria including a Pseudomonas cepacia invarious chlorinated compounds including chlorotoluene. Clark, R. R.,Chian, E. S. K. and Griffin, R. A., (1970) Applied & EnvironmentalMicrobiology 680-685 discuss the Degradation of PolychlorinatedBiphenyls by Mixed Microbial Cultures and conclude the higher thechlorine content the more difficult it is to biodegrade. Furukawa, K.Tonomura, K. and Kamibayashi, A., (1978) Applied & EnvironmentalMicrobiology, 35 No. 2, 223-227, "Effect of Chlorine Substitution on theBiodegradability of Polychlorinated Biphenyls", show the effect ofchlorine substitution on biodegradability of polychlorinated biphenyls.Shapiro, J. A. et al, (1980) in "Perspectives for Genetic Engineering ofHydrocarbon Oxidizing Bacteria" published in Trends in the Biology ofFermentation for Fuels and Chemicals, Brookhaven National Laboratory,Dec. 7- 11, 1980 gives perspectives for genetic engineering ofhydrocarbon oxidizing bacteria. It has been widely believed thatdechlorination of chlorinated aromatic compounds only occurred afterdearomization of the aromatic ring. For example, after meta fision ofthe ring, the ability of whole cell suspension of Methylosinustrichosporium to dechlorinate chlorotoluenes without ring cleavage hasbeen demonstrated. See Higgins, I. J. Hammond, R. C., Sariaslani, F. S.,Best, D., Davies, M. M., Tryhorne, S. C. and Taylor, M. F. (1979),"Biotransformation of Hydrocarbons and Related Compounds by WholeOrganisms Suspension of Methane-Grown Methylosinus trichosporium OB36",Biochem. Biophys. Res. Commun., 89, 671-677. Products of the reactionincluded benzyl alcohol, benzyl epoxide and methyl benzyl alcohol.Dechlorinating ability of M. trichosporium was attributed to theactivity of the methane monooxygenase system of the organism. These arethe more pertinent examples of the scientific publications available asbackground on the Microbial degradation of organic compounds.

Notwithstanding all the effort, as represented by the scientificpublications in this area of technology, there has been no practicalapplication of these technologies in cleaning up the ecosphere.Furthermore, it has been suggested that because halogenated compoundsare not generally found in nature, microorganisms have not evolved whichpossess efficient enzyme systems or genes which express themselves forthe degradation of such chemicals; see Chatterjee, D. K., Kellogg, S.T., Furukawa, K., Kilbane, J. J., Chakrabarty, A. M., "GeneticApproaches to the Problems of Toxic Chemical Pollution", Third ClevelandSymposium on Macromolecules, 1981. Chakrabarty disclosed a technique forartificially inducing the biodegradability of 2,4,5 trichlorophenylacetic acid (2,4,5 T) by gradually exposing bacteria to increasedconcentrations of the chemical over the course of about one year; seeChatterjee, D. K., Kellog, S. T., Eatkins, D. R. and Chakrabarty, A. M.in Levy, S., Clowes, R. and Koenig, E. (Eds.), "Molecular Biology,Pathogenicity and Ecology of Bacterial Plasmids", Plenum PublishingCorp., N.Y., 1981, pp. 519-528.

Contrary to these teachings, and much to our surprise, we have foundthat microorganisms present in the locus of concentrated deposits ofhalo-organic chemicals have not only managed to stay alive but haveadapted themselves to grow and multiply on the halo-organic e.g.,chlorinated hydrocarbons in the landfill as their sole source of carbonand energy.

Although we do not wish to be confined to any theory as to why thisphenomena exists, we offer the following possibilities, provided theyare not construed as limiting our invention and discoveries, except asexpressly contained in the appended claims.

Prior to chemical wastes being accumulated in a given landfill the soiland surrounding environment was populated with bacteria that used thenormal hydrocarbon material in the soil as their source of carbon andenergy. As the obnoxious chlorinated organics and other wastes weredeposited on them, all but the strongest were killed off. Per CharlesDarwin's theory of survival of the fittest including the process ofevolution which includes isolation, speciation, mutation and otherprocesses and mechanisms, conjugation into other bacteria whether of thesame strain, species, genera or different involved in the process ofnatural selection certain of these bacteria adapted their metabolisms tobreak the carbon-chlorine bond causing the formation of more easilymetabolized materials, e.g. catechols or further metabolized derivativecompounds, haloaliphatic compounds, which are more in line with thesubstrates the bacteria have been accustomed to metabolizing.

Another possibility is that the bacteria in the soil always possessedgenes in their DNA which were capable of metabolizing chlorinatedorganics. These genes may have been active and expressed in theancestors of the organisms when salt and chlorine may have been moreconcentrated and abundant in their sites. But in recent times, thebacteria never had to use them, i.e., such genes were not expressedbecause there were easier metabolized carbon material on their sites forthem to thrive on. This theory supports the fact that the lowconcentrations of chlorinated organics in the ecosphere due to pesticideapplications are not concentrated enough to cause the bacteria to adaptto metabolizing them, thus, the persistance of these chemicals in thesoil and ecosphere and their nonbiodegradability.

No matter what the theory, we have found that bacteria capable ofbiodegrading a waste are created at the locus of the waste when there isa significant concentration of the waste to the exclusion of othersources of carbon for the bacteria to feed on and when enough time haselapsed thereby allowing for the mutations in the bacteria which allowfor their biodegradation to be continuously expressed from onegeneration to the next.

DESCRIPTION OF THE INVENTION

Samples of soil and leachate were recently obtained from a landfill siteof the Hooker Chemical and Plastics Corp. in Niagara Falls, N.Y. whichhad been used for disposal of chlorinated organic wastes during theperiod 1955 to 1975. These samples were utilized in enrichmentexperiments and were found to contain microorganisms that were able todissimilate 2-chlorotoluene, 3-chlorotoluene, 2,6-dichlorotoluene,3,4-dichlorotoluene, 2,4-dichlorobenzoate and 3,4-dichlorobenzoate assole carbon and energy sources for growth. The identity of the bacteriathat degrade the chloroaromatic compounds was established as Pseudomonasspecies and are specifically designated respectively as followsHCI(2CT), HCIV(3CT), HCV(2,6-DCT)-2, HCV(3,4-DCT)-5, HCV(2,4-DCB),HCV(3,4-DCB), HCV(2,6-DCT)-3, HCV(3,4-DCT)-7, HCV(2,6-DCT).HCV(2,6-DCT)-2, and HCV(2,6-DCT)-3 have been further identified asPseudomonas cepacia and all strains will be identified as Pseudomonascepacia var., niagarous.

The processes of sampling, enrichment, isolation, separation,purification and application employed will be given in the exampleswhich follow. Further disclosure and identification of the bacteriafollows.

Cultures of the Pseudomonas species have been deposited with theAmerican Type Culture Collection, 12301 Parkway Drive, Rockville, Md.20852. The microorganisms have been given the following identifying ATCCnumbers.

    ______________________________________                                        Strain Designation                                                                            ATCC Number                                                   ______________________________________                                        HCI(2 CT)       ATCC-31945                                                    HCIV(3 CT)      ATCC-31941                                                    HCV(2,4 DCB)    ATCC-31942                                                    HCV(3,4 DCB)    ATCC-31940                                                    HCV(2,6 DCT)-2  ATCC-31943                                                    HCV(2,6 DCT)-3  ATCC-31944                                                    HCV(3,4 DCT)-5  ATCC-31939                                                    ______________________________________                                    

The organism shall be made permanently available to the public inaccordance with the Apr. 29, 1971 Commissioner's notice appearing at8860G638.

THE FIGURE AND THE TABLES

FIG. 1 shows the Agarose gel electrophoresis analysis of cesium chlorideethidium bromide purified DNA preparations derived from parentalrecipient transformant and transconjugant strains as will be discussedlater in the examples.

Table I gives the Morphological, Cultural and Physiological propertiesof a specific Pseudomonas cepacia var., niagarous HCV (2,6 DCT)-2. Thephysiological properties in Table I were determined by the API 20ESystem which is a standardized miniaturized version of conventionalprocedures for the identification of Enterobacteriaceae and otherGram-negative bacteria. Analytab Products, 200 Express Street,Plainview, N.Y. 11803.

The microorganism Pseudomonas cepacia is a Gram negative bacteriumhaving the following characteristics: rods which are very short andplump with the following usual dimensions--0.5 μm by 1.5-4 μm.

Table II gives the hydrocarbon utilization at 300 ppm of some of thePseudomonas cepacia var., niagarous of this invention. After primaryisolation and purification utilizing one substrate as the carbon source,the microorganisms were tested for growth on other substrates at 300 and1000 ppm. The growth was monitored on solid media vs. nutrient agar ascontrol.

Table III gives the hydrocarbon utilization of the Pseudomonas cepaciavar., niagarous of 1000 ppm.

Table IV gives the antibiotic resistances of some of the Pseudomonascepacia var., niagarous.

Table V gives the substrate utilization profile for transformants andconjugants given in Example I for PAO 2178 (pRO 63) which identificationis in accordance with the National Plasmid Registry Stanford UniversityMedical School, Palo Alto, Calif.

TABLE I MORPHOLOGICAL/CULTURAL/PHYSIOLOGICAL PROPERTIES PSEUDOMONASCEPACIA (STRAIN HCV (2,6 DCT)-2 ATCC-31943

A. Morphological Properties

1. Small rods

2. Gram (-) negative

3. No spores

B. Cultural Properties

1. Growth at ambient to 42° C.

2. Growth on mineral salts and carbon source

3. Yellow pigment @37° C. on nutrient media

4. Glossy smooth, entire colonies on nutrient media

C. Physiological Properties

1. -galactosides+(hydrol of ONPG)

2. Arginine decarboxylase (-)

3. Lysine decarboxylase (-)

4. Citrate (+)

5. H₂ S produced from Thiosulfate (+)

6. Ammonia not produced from urea (-)

7. Gelatinase (+)

8. Carbohydrate utilization

(a) Inositol (-)

(b) Rhamnose (-)

(c) Melibiose (-)

(d) Amygdalose (-)

(e) Arabinose (-)

(f) Glucose (+)

(g) Mannitol (+)

(h) Sorbitol (+)

(i) Sucrose (+)

9. Oxidase (+)

10. Nitrate reduced to nitrite (+)

                                      TABLE II                                    __________________________________________________________________________    HYDROCARBON UTILIZATION AT 300 PPM SUBSTRATE CONCENTRATION*                   Compound   HCI 2CT                                                                            HCIV 3CT                                                                            HCV 2,4DCB                                                                            HCV 3,4DCB                                                                            HCV 3,4DCT                                                                            HCV 2,6DCT-2                    __________________________________________________________________________    2 Chlorotoluene                                                                          2.3  2.3   2.3     2.3     2.3     2.3                             3 Chlorotoluene                                                                          3    3     3       3       3       3                               3,4 Dichlorotoluene                                                                      3    3     3       3       3       3                               2,6 Dichlorotoluene                                                                      2.3  2     2.3     2.3     2.3     2                               Benzoate   4    0     4       4       4       4                               4 Chlorobenzoate                                                                         2.3  1.7   2       2       2       2.3                             2,4 Dichlorobenzoate                                                                     3    2     3       3       3       3                               2,4 D      3    2     3       3       3       3                               2,4,5 T    2    2.5   2       2       1       2                               __________________________________________________________________________     *Growth on substrate vs. nutrient agar scored from 1 to 4 with 4              designating highest growth rate.                                         

                                      TABLE III                                   __________________________________________________________________________    HYDROCARBON UTILIZATION AT 1000 PPM SUBSTRATE CONCENTRATION                   Compound   HCI 2CT                                                                            HCIV 3CT                                                                            HCV 2,4DCB                                                                            HCV 3,4DCB                                                                            HCV 3,4DCT                                                                            HCV 2,6DCT-2                    __________________________________________________________________________    2 Chlorotoluene                                                                          3    2     3       3       3       2.5                             3 Chlorotoluene                                                                          3    2     3       3       3       2.5                             3,4 Dichlorotoluene                                                                      3    2     3       3       3       2.5                             2,6 Dichlorotoluene                                                                      2.5  2     3       3       3       2                               Benzoate   4    0     4       4       4       4                               4 Chlorobenzoate                                                                         3    0     3       3       3       3                               2,4 Dichlorobenzoate                                                                     4    3     4       4       4       4                               2,4 D      3    1.5   3       3       3       3                               2,4,5 T    2.5  1     2.5     2.5     2.5     2.5                             __________________________________________________________________________

                                      TABLE IV                                    __________________________________________________________________________    ANTIBIOTIC SUSCEPTIBILITY*                                                    (Numbers in parentheses are concentrations in millicentigrams)                __________________________________________________________________________            TETRA-                                                                              NALIDIXIC                                 CARBENI-                      CYCLINE                                                                             ACID   AMPICILLIN                                                                            GENTAMICIN                                                                             PENICILLIN                                                                            ERYTHROMYCIN                                                                            CILLIN                        (30)  (30)   (10)    (10)     (15)    (50)      (50)                  __________________________________________________________________________    HCI 2CT R     I      R       S        R       R         NA                    HCIV 3CT                                                                              S     S      R       S        R       R         R                     HCV 2,4DCB                                                                            R     R      R       S        R       R         R                     HCV 3,4DCB                                                                            R     R      R       S        R       R         R                     HCV 3,4DCT                                                                            I     S      R       S        R       R         R                     HCV 2,6DCT-2                                                                          S     S      R       S        R       R         S                     __________________________________________________________________________                      CARBENI-                                                                             CHLORAM-     STREPTO-                                                  CILLIN PHENICIL                                                                             COLISTIN                                                                            MYCIN  KANAKMYCIN                                                                             AUREOMYCIN                                (100)  (30)   (10)  (10)   (30)     (30)                    __________________________________________________________________________              HCI 2CT R      R      S     S      S        NA                                HCIV 3CT                                                                              R      I      S     R      S        S                                 HCV 2,4DCB                                                                            R      R      S     I      S        S                                 HCV 3,4DCB                                                                            R      R      S     I      S        S                                 HCV 3,4DCT                                                                            R      R      S     I      S        S                                 HCV 2,6DCT-2                                                                          S      S      S     I      I        S                       __________________________________________________________________________     *Antibiotic Susceptibility scored by the method of Kerby and Bauer, Am. J     Clinical Path., 45, 493 (1966).                                          

                                      TABLE V                                     __________________________________________________________________________    SUBSTRATE UTILIZATION PROPERTIES OF DERIVED STRAINS                                                  TRANSFORMANTS       TRANSCONJUGANTS                               PAO         PAO       PAO       PAO       PAO                      Compound   2178                                                                             HCV(2,6 DCT)-2                                                                         2178(2,6 DCT-2)-1                                                                       217(2,6 DCT-2)-2                                                                        2178(2,6 DCT-2)-3                                                                       2178(2,6                 __________________________________________________________________________                                                         DCT-2)-4                 2 Chlorotoluene                                                                          -  +        +         +         +         +                        3 Chlorotoluene                                                                          -  +        +         +         +         +                        4 Chlorotoluene                                                                          -  +        +         +         +         +                        2,6 Dichlorotoluene                                                                      -  +        +         +         +         +                        3,4 Dichlorotoluene                                                                      -  +        +         +         +         +                        2 Chlorobenzoate                                                                         -  +        +         +         +         +                        3 Chlorobenzoate                                                                         -  -        +         +         +         +                        4 Chlorobenzoate                                                                         -  -        +         +         +         +                        2,4 Dichlorobenzoate                                                                     -  +        +         +         +         +                        3,4 Dichlorobenzoate                                                                     -  +        +         +         +         +                        p-Toluic Acid                                                                            -  -        +         +         +         +                        Toluene    -  -        +         +         +         +                        __________________________________________________________________________     Volatile carbon sources were incorporated directly in the medium as well      as in the vapor phase in a sealed container.                                  + = Growth                                                                    - = No Growth                                                            

THE EXAMPLES

The following examples are given to further describe our inventionhowever,, they are given for illustrative purposes only and are notincluded to limit the scope of our invention except as defined in theappended claims.

EXAMPLE I Collection

Soil samples were collected during the month of May from portions of alandfill site at a depth of about above 8 to 12 inches to the top of thesoil in Niagara Falls, N.Y. at a place where there was a highconcentration of contaminants and where the odor of halogenatedchemicals in the soil was self-evident. The landfill has been used forobnoxious organic wastes including chlorinated organics for a period ofover 20 years.

Enrichment

Approximately 1 gram of soil was suspended in 25 ml of minimal saltsmedia of the following composition; contained per liter: 40 ml of Na₂HPO₄ +KH₂ PO₄ buffer (pH 6.8); 20 ml of Hutner's vitamin-free mineralbase and 1.0 g of (NH₄)₂ SO₄. The medium contained 0.2% of L Tryptophanand 0.05% of Difco (Difco Laboratories, Detroit, Mich.) yeast extract,according to the prior art Lichstein, H. C. and Oginsky, E. L. inExperimental Microbial Physiology, W. H. Freeman & Company (1965). Theenrichment culture was statically incubated in shallow culture for 72hours at 25° C.

Isolation & Purification

After incubation, 0.1 ml of the enrichment broth was spread over thesurface of solid minimal salts media with a bent glass rod and theplates incubated at 25° C. The solid media also contained 0.2% of aspecific haloaromatic carbon source. Several serial isolations werecarried out by removing colonies with a wire loop and streaking over thesurface of fresh solid media containing a specific haloaromatic carbonsource. In this way purified isolates were obtained.

After primary isolation and purification utilizing one substrate as thecarbon source, the microorganisms were then tested on solid media forgrowth on other chlorotoluenes and chlorobenzoate compounds as shown inTable II and III.

Preperation of DNA

Cells from ATCC 31943 which contain plasmids pRO 4.7, pRO 31, and pRO 54were grown on the surface of nutrient agar plates overnight at 23° C.The cell crops were removed from the nutrient agar plates washed andresuspended at high density in buffered 25% sucrose (pH 8.0) asdescribed by Hanson and Olsen, J. Bacteriology, 135, 227 (1978). Allsubsequent mixing was done by slow, gentle inversion. To lyse cells, weadded lysozyme and ethylenediaminetetraacetate, and then sodium dodecylsulphate (SDS) to 4% final concentration. Eight repeated cycles of heatpulse and mixed produced a clear, viscous lysate. DNA was denatured atpH 12.1-12.3 by adding 3M NaOH and mixing for 3 min. at roomtemperature. Then tris(hydroxymethyl)aminomethane (pH 7.0) was added toreturn the pH below 9.0. We added SDS to 4% final concentration, NaCl to1.0M and mixed by 20 inversions; after 6 hours at 4° C., thesalt-precipitated chromosome-membrane complexes were pelleted bycentrifugation at 17,000 g (4° C., 30 min.). The supernatant was mixedwith polyethylene glycol 6000 to 10% concentration. After 6 hours at 4°C., the tubes were centrifuged at 700 g (4° C., 5 min.). Resuspension ofthe resulting pellets in 0.15 ml cold buffer gave plasmid-enriched DNAsolution. Agarose slab gel electrophoresis, for 3 hours at 100 voltsthrough 0.7% agarose (wt./vol.), was carried out using the method ofMeyers (Meyers, J. A., Sanchez, Dr. Elwell, L. P. and Falkow, S.,Journal of Bact., 127, 1529 (1978). Each well contained 25 μl ofplasmid-enriched DNA solution mixed with 10 μl Meyers tracking dye. Gelswere stained with ethidium bromide solution and visualized on anultraviolet transilluminator.

FIG. 1 shows a typical Agarose slab gel electrophoresis which isexplained in a later part of the disclosure.

Transformation

A portion (25 ml) of TN broth (tryptone, yeast extract, glycose andsalt) was inoculated with cells from a nutrient agar plate grownovernight and incubated at 37° C. in a shaker until the optical density(425 μm) was 1.0. The mixture was centrifuged cold at 10 rpm for 18minutes and the cells separated from the supernatant and resuspended in10 ml of 0.15M MgCl₂ and allowed to stand for 5 minutes in ice. Themixture was centrifuged again and resuspended as above and held on icefor 20 minutes. The mixture was centrifuged and resuspended as above athird time and 0.2 ml of the resuspended cells added to prepared DNA(10-50 μl) in a cold centrifuge tube and held on ice for 60 minutes.Heat pulse was applied for 2 minutes at 37° C. and then chilled.Finally, 0.5 ml TN broth at room temperature was added and the mixturestaticly incubated 1-2.5 hours at 37° C.

This recipient strain, PAO 2178, (see Royle, P. L., Matsumoto, H. andHolloway, B. W., J. Bacteriol., 145, 145 (1981) requires methionine andis a catechol-1,2-oxygenase mutant and consequently is unable to degradebenzoate and chloroaromatic compounds. However, when PAO 2178 wastransformed with CsCl purified plasmid DNA prepaed from HCV(2,6-DCT)-2,the ability to utilize 2,6-dichlorotoluene was introduced into PAO 2178at a frequency of approximately 1.9×10⁵ transformants/μg DNA. Suchtransformants were able to metabolize all of the chlorotoluene andchlorobenzoate compounds examined, unlike the genetic donor, HCV(2,6DCT), which did not utilize 3-chlorobenzoate or 4-chlorobenzoate. Thesubstrate utilization profile for the transformants, designated PAO2178(2,6-DCT-2)-1 and -2, and is also identified herein as PAO 2178(pRO63), said identification being in accordance with the National PlasmidRegistry Stanford University Medical School is shown in Table V.However, this and other transformants (not shown) still requiredmethionine for growth and were unable to utilize benzoate.

Conjugation

Conjugal mating experiments were done using PAO 2178 as the recipientbacterial strain. Strain PAO 2178 was a mutant of PAO1_(c) as describedby Royle, Matsumoto and Holloway. Minimal salts medium and complexmedium were prepared as described by Olsen, R. H. and Hansen, J., J.Bacteriol., 123, 28 (1975) and J. Bacteriol., 125, 837 (1976). Whennutritional selection against auxotrophic donors was done, amino acidrequirements were satisfied by the addition of these components to afinal concentration of 20 μg/ml.

All matings were done in TN broth medium. For this, TN broth medium wasinoculated with overnight growth from TN agar. These broth cultures wereincubated for 3 hours with agitation at 37° C. Inoculation was adjustedto result in approximately 10⁸ cells per ml of TN broth culture after 3hours of growth. Donor and recipient cells were mixed 1:1 and incubatedat 37° C. for 2 hours. Mating mixtures were centrifuged at ambienttemperature, and cell pellets were suspended to 1/10 the original volumeof 0.01M phosphate buffer (pH 7.0). Cell suspensions were diluted andplated onto mineral salts medium supplemented with the nutrients isspecified by the recipient and a haloaromatic compound whose utilizationrequired by the doner plasmid. Plates were incubated for 48 hours at 23°C. Transconjugants were purified by picking colonies into liquidsuspension, followed by streaking out for single-colony isolation onsolid medium identical to that used for their primary isolation.

The transconjugants obtained were able to metabolize all of thechlorotoluene and chlorobenzoate compounds examined, as was the case forthe genetic donor in these conjugation experiments, HCV(2,6-DCT)-2. Inaddition, they also utilized 3- and 4-chlorobenzoate, compounds notutilized by the doner. The substrate utilization profile for thesetransconjugants, designated PAO 2178(2,6-DCT-2)-3 and -4 is shown inTable V.

Plasmid DNA was extracted from HCV(2,6-DCT)-2 and a transformant and atransconjugant derived from HCV(2,6-DCT)-2, as described earlier. TheseDNA preparations were purified in cesium chloride-ethidium bromidegradients and subjected to slab agarose gel electrophoresis. In FIG. 1,file A, plasmid DNA from the parenteral strain, HCV(2,6-DCT)-2, is shownand it contained plasmids of 4.7, 31 and 54 Mdaltons in molecular size(plasmid size was determined previously using appropriate standards).File B contains the recipient strain PAO 2178. The results in File Bdemonstrates that the recipient strain does not harbor residentplasmids. File C, shows plasmid DNA extracted from a transformant,strain PAO 2178 (2,6-DCT-2)-1. File D, shows plasmid DNA from atransconjugant strain PAO 2178 (2,6-DCT-2)-3. As shown in files C and D,the plasmid band from either the transformant or transconjugant isapproximately 63 Mdaltons in molecular size.

The molecular sizes of plasmids from either transconjugant ortransformant strains were similar but larger than those observed in thedonor strain HCV(2,6-DCT)-2. This result was unexpected since twodifferent genetic techniques were utilized to transfer the plasmids tothe recipient, PAO 2178. The explanation for this is the occasionalrandom formation of a fusion plasmid in the donor bacterial culture,which contains the 4.7 and 54 Mdalton plasmids present usuallyindependently in a donor bacterium. These fusion plasmids have beenuniquely selected in the genetic transfer experiments because one of thedonor plasmids contains a replicator functional in strain PAO 2178 andthe other donor plasmid component contains a nonfunctional replicatorbut has the genes which encode for the degradative activities.Therefore, maintenance of the degradative activity, in this strain,requires the maintenance of the replicator contained in the 4.7 Mdaltonplasmid.

The PAO 2178 strain acquired the ability to utilize variouschloroaromatic compounds through transformation or conjugation withHCV(2,6-DCT)-2. The results demonstrate that PAO 2178 acquired the genesnecessary for chloroaromatic compound utilization from a transmissibleplasmid. Furthermore, the strains acquired through conjugation ortransformation showed the ability to use two compounds not degraded bythe donor (i.e., 3-chlorobenzoate and 4-chlorobenzoate. This reflectsthe supportive metabolism present in PAO 2178 not present inHCV(2,6-DCT)-2 for the complete dissimilation of these compounds.

Application

To approximately a kilo of each three clean soils are added 1 gram of3,4-dichlorobenzoate, 3-chlorobenzoate and 2,6-dichlorotoluene toproduce soil samples contaminated to 1000 ppm. Fifty ml of liquid mediaculture containing HCV(2,6-DCT)-2 and HCV(3,4 DCB) are added to the soiland the mixture incubated at 25° C. Five gram samples of soil are takendaily and analyzed for the substrate.

Five grams of soil are weighed into a 50 ml erlenmeyer flask containing10 ml of distilled water. The pH of the soil suspension is adjusted to4.0 with dilute H₂ SO₄ and extracted with 3 10 ml portions of methylenechloride. The methylene chloride extracts are transferred to a 50 mlvolumetric and made up to volume. The methylene chloride solution isanalyzed a 254 μm using a Beckman Lamda 3 UV/VIS spectrometer.

Monitoring the utilization of the chloroaromatic substrates shows that90% of the chloroaromatic substrates are decomposed within 1 week.

In order that the scope and breadth of the process of our invention forbiodegrading the contaminants in the ecosphere back into the naturalcarbon cycle may be more readily understood, the following additionaldisclosures are made in connection with it.

EXAMPLE II

A sample of contaminated air is obtained in a standard air bag or filtersampling device. The airborne bacteria in the sample which have adaptedthemselves to metabolize the organic contaminants in the contaminatedair back into the natural carbon cycle are enriched, isolated, purified,identified, and produced substantially in accordance with the proceduresgiven in Example No. 1 on HCV (2,6-DCT-2). In this case, the bacteriaare applied to the contaminated air in a closed system by knowntechniques. By periodic monitoring of samples from the locus of thecontaminated air, one will find that there will be a reduction in thedensity of the contaminants.

EXAMPLE III

Leachate from chemical waste landfill drainage is placed in a 500 gallonglass lined pressure reactor, agitated and heated to a temperature ofabout 30° C. To this reactor is added 2000 parts per million of each ofthe Pseudomonas strains isolated as in Example I. After 38 hours, thepressure in the reactor will increase up to about 2 atmospheres. Thepressure is released through a valve in the reactor. The vented gasesare analyzed and found to be mainly carbon dioxide. The remainingcontents in the reactor are analyzed and are found to be carbonatedsalt-water. The concentrations of the noxious toxic chemicals arereduced.

EXAMPLE IV

A new chemical containing 12 chlorine, 12 carbon and 12 hydrogen atomsis made by the Diels-Alder reaction using the appropriate materials. Thechemical is found to be useful in large tonnage industrial applicationsbut its residues and by-products which comprise 25% by weight of thedesired product are toxic, obnoxious and not biodegradable simplybecause this material is a new composition of matter never existant innature. Accordingly, bacteria have never been given a chance to adapt tobiodegrade it. In accordance with this invention, the wastes aredisposed of in a landfill known to house bacteria not capable ofbiodegrading the wastes. Samples of the soil are taken at monthlyintervals and after 10 months, a bacteria is isolated capable ofbiodegrading the waste. These bacteria are enriched in accordance withExample I and produced in large quantity for storage. The chemicalprocess equipment in the manufacturing plant for the new composition ofmatter is adapted to contain an add-on reactor along the lines given inExample III, so that the by-product and effluent streams may be treatedby the enriched strains of bacteria which have been recovered from thelocus of the landfill which are capable of biodegrading the obnoxiouswastes in the plant effluents. In this manner, the process is madeecologically safe and less harmful because of the smaller amount ofobjectional effluents from this chemical process.

In order that this invention may be more readily understood in greaterdetail, the following guidelines are given using primarily oneembodiment of our invention namely, the biodegradation of a longestablished chemical landfill containing obnoxious halogenated organicchemicals. However, it should be understood that many of theseguidelines are useful in other applications such as collecting bacteriafrom aqueous environments including lake beds, streams and sediments orfrom the atmosphere.

In accordance with our invention, bacteria are isolated from the soil ofthe landfill which soil sample is selected in areas of highconcentrations of organic chemical contamination. The continued exposureof bacteria to the high concentrations of contaminants in theseenvironments increases the tolerance of such strains to the contaminantsby the process of natural selection and adaptation to the point whereover the course of time only strains of baccteria or microbes which cansurvive such an environment are present. Thus, we employ naturallymutated bacteria which have adapted to live on the chlorinatedhydrocarbons as their sole source of carbon and energy as the startingmaterial in our process. It is a further purpose of this invention toprovide a process for maintaining and controlling the natural process ofselection to produce bacteria with the specialized and desirable wastedisposal capabilities. We have found that when our new strains ofbacteria are inoculated or otherwise reintroduced to the contaminatedenvironment, particularly if their favorite substrates are either absentor only present in low concentration, they multiply rapidly bymetabolizing the halogenated organics to carbon dioxide, water and salt.On the other hand, if the new strains of bacteria are applied to a lowconcentration contaminated environment containing more favoritesubstrates, then their ability to biodegrade the chlorinated organics isreduced, if not terminated, or rendered latent or impotent; thus theyrevert to type unless continually forced to use the waste contaminantsas their source of carbon. Employing more favored substrates is one wayof controlling the population or even the existance of the bacteria inthe location it is used in. Other methods of controlling or destroyingthe bacteria, if their growth and population exceed the desired limit isto use a non-resistant antibiotic or other biocide.

The collecting of the sample to be enriched in accordance with ourinvention should be made at a site containing a high concentration ofcontaminants whether it be in the soil, water or air. The age of locusof contaminated area should be old enough to have allowed for manygenerations of bacteria to grow so that the bacteria have developedgenetically the ability to utilize and degrade the contaminant.

Since the disposal of chemicals in landfills usually was done on achronological basis and because similar chemicals were usually placed inthe same location in the landfill, it may become necessary to takesamples of the soil in the landfill from various areas in order to findthe microorganisms or bacteria capable of degrading the variouschemicals in the landfill. For example, if the northeast quadrant of thelandfill was filled with residues and wastes resulting from 2,4,5 T(2,4,5 Trichlorophenol), and, if the southwest quadrant was filled withresidues and wastes from the manufacture of chlorotoluenes, then samplesof soil from each quadrant (or other measure of the landfill) should beobtained and processed in accordance with this invention, in order tofind the best variety of microorganisms or bacteria capable of beingupgraded and enhanced, for application in the degradation of the wastesand residues.

Another factor that is important to consider in sampling from thelandfill is whether or not the landfill has been "seasoned", that is,has gone through the four seasons of a year so that the microorganismsor bacteria had mutated in a way that allows them to degrade thechemical and other wastes and is also adapted to survive the vigoroustemperature and other conditions caused by the extremes of summer heatand winter cold. This is especially important in the northern climatessuch as in Niagara Falls, N.Y. where temperatures in the summertime canreach 100° F., and in the wintertime below -30° F., and snowfall canexceed 144 or more inches in one winter season. Thus, the atmospheric,weather and other related conditions surrounding the environment of thelandfill should be taken into consideration when sampling for bacteria.

Still another factor that should be considered is the depth of soil oneshould go to, in extracting the samples. Since the microorganisms orbacteria of this invention are aerobic, best strains which are useful inthis invention are obtained from the top layer perhaps 8-12 inches deepof the landfill. This is not to preclude sampling for microorganisms orbacteria at depths in the landfill greater than 8-12 inches. Forexample, soil conditions and composition will effect permeation of airto greater depths. In addition, anaerobic bacteria can be found at stillgreater depths. There are known landfills which have exceeded 50 ft indepth. Accordingly, sampling at such depths for microorganisms andbacteria are embraced within the concepts of this invention.

Any one of a number of techniques may be used to enrich the bacterialstrains in accordance with our invention. For example, instead of theprocedure used in Example I, one may employ standard minimal nutrientmedia and an appropriate carbon source such as given in Hartmann, J.,Reineke, W., and Knackmuss, N. J., Applied and EnvironmentalMicrobiology, 37, 421 (1979).

The separation, isolation and purification may be done by any one orcombinations of the following techniques. The standard microbiologicalprocedure of serial dilution can be applied to enriched mixtures ofbacteria to separate, isolate and purify the strains in the enrichmentmedia. Other techniques such as repeated plating on non-inhibitory mediaand single cell isolations can be used.

Bacteria isolated and described in this way may in some instances serveas a source of critical genetic information for haloaromatic compounddegradation that may be acquired by yet other bacteria by any one of ora combination of genetic processes utilized by bacteria for theformation of genetic hybrids. These genetic processes includeconjugation and transformation as described herein and also the geneticprocess of transduction. Bacterial transduction is characterized by thetransport of genes from a doner bacterium to a recipient bacterium byviruses grown on the doner and later infecting the recipient bacterium.Still another process called cell fusion may be utilized for geneticexchange by which environmental conditions are established which causetwo bacterial cells, a doner and a recipient, to become one cell withcommon genetic material and cellular cytoplasm. The daughter cellsproduced when these fused cells reproduce now contain genesrepresentative of both participants in the initial cell fusion.

Various other methods of applying the bacteria to the contaminants to bedisposed of may be used. For example, the microorganisms isolated as inExample I are injected along with nutrient media and oxygen intochemical waste landfill. The organisms utilize the waste stored in thelandfill as sole source of carbon and energy thus destroying thecontents of the landfill.

Soil contaminated by a chemical spill is inoculated with microorganismsisolated as in Example I and the soil subsequently cultivated tooxygenate the soil. Cultivation continues for about 1 week until thechemical residue is reduced to nonhazardous levels.

Another mode of application involves employing bacterial strains orcultures produced in accordance with this invention which have beencollected from a room housing a chemical plant for manufacture ofchlorinated hydrocarbons, having several parts per million ofchlorinated contaminants in the air. The bacterial strains and culturesmade in accordance with this invention are desposited on an air filtersystem and the contaminated air from the plant room circulated throughthe filter system to produce air less contaminated with the chlorinatedmaterials.

The work done herein was all done in conformity with physical andbiological containment requirements specified in the Guidelinespublished by the National Institute of Health, Washington, D.C. UnitedStates of America.

Although our invention has been described using specific examples andcertain preferred embodiments thereof, we do not intend that ourinvention be limited in scope except as expressly defined in theappended claims.

We claim:
 1. A process for microbial degradation of halogenated organicchemical waste which comprises applying to the locus of said halogenatedorganic chemical waste strains of microorganisms capable of degradingsaid halogenated organic chemicals to carbon dioxide, water, and a saltsaid strains being a member of the group consisting of Pseudomonascepacia var. niagarous ATCC 31945, ATCC 31941, ATCC 31942, ATCC 31940,ATCC 31943, ATCC 31944, ATCC 31939, and mutants thereof and monitoringremoval of the contaminants from the locus of the application.
 2. Aprocess in accordance with claim 1 wherein the halogenated organicchemical wastes have been stored in a landfill and said strains ofmicroorganisms have been taken from said landfill.
 3. A process inaccordance with claim 1 wherein the locus of the contaminants to bedegraded is a leachate removed from a landfill and said strains ofmicroorganisms have been taken from a landfill.
 4. A process inaccordance with claim 1 wherein the locus of the contaminants to bedegraded is soil containing halogenated organic chemical wastes, thesoil being cultivated in the presence of oxygen or air with strains ofmicroorganisms taken from a soil site and combined with nutrient.
 5. Aprocess in accordance with claim 4 wherein the locus to be degraded is alandfill.
 6. A process in accordance with claim 1 wherein thehalogenated organic chemical wastes are predominantly chlorinatedorganic chemicals.
 7. A process in accordance with claim 5 wherein thechlorinated organic chemicals contain chlorotoluenes and metabolicderivative compounds.
 8. A process in accordance with claim 1 whereinthe strain of microorganism employed is ATCC 31945, and mutants thereof.9. A process in accordance with claim 1 wherein the strain ofmicroorganism employed is ATCC 31941, and mutants thereof.
 10. A processin accordance with claim 1 wherein the strain of microorganism employedis ATCC 31942, and mutants thereof.
 11. A process in accordance withclaim 1 wherein the strain of microorganism employed is ATCC 31940, andmutants thereof.
 12. A process in accordance with claim 1 wherein thestrain of microorganism employed is ATCC 31943, and mutants thereof. 13.A process in accordance with claim 1 wherein the strain of microorganismemployed is ATCC 31944, and mutants thereof.
 14. A process in accordancewith claim 1 wherein the strain of microorganism employed is ATCC 31939,and mutants thereof.
 15. A process in accordance with claim 1 whereinthe strain of microorganism employed contains a plasmid selected fromthe group consisting of of pRO 4.7, pRO 31 and pRO 54 having utility indecomposing halogenated organic chemicals to innocuous materials, saidplasmid characterized by having a molecular weight of between 4.7 and 54megadaltons and being capable of fusing with other plasmids of highermolecular weight.
 16. A process in accordance with claim 15 wherein thestrain of microorganism employed contains pRO 4.7.
 17. A process inaccordance with claim 15 wherein the strain of microorganism employedcontains pRO
 31. 18. A process in accordance with claim 15 wherein thestrain of microorganism employed contains pRO
 54. 19. A process inaccordance with claim 15 wherein the plasmid in the strain ofmicroorganism employed is a hybrid plasmid containing pRO 4.7, pRO 31,and pRO 54.